U.S. patent application number 10/840425 was filed with the patent office on 2005-03-03 for methods for sterilizing preparations of digestive enzymes.
This patent application is currently assigned to Clearant Inc.. Invention is credited to Burgess, Wilson, Drohan, William N., Griko, Yuri, MacPhee, Martin J., Mann, David M..
Application Number | 20050047958 10/840425 |
Document ID | / |
Family ID | 25478847 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050047958 |
Kind Code |
A1 |
Mann, David M. ; et
al. |
March 3, 2005 |
Methods for sterilizing preparations of digestive enzymes
Abstract
Methods are disclosed for sterilizing preparations of digestive
enzymes to reduce the level of one or more active biological
contaminants or pathogens therein, such as viruses, bacteria
(including inter- and intracellular bacteria, such as mycoplasmas,
ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds,
fungi, prions or similar agents responsible, alone or in
combination, for TSEs and/or single or multicellular parasites.
These methods involve sterilizing preparations of digestive
enzymes, such as trypsin, .alpha.-galactosidase and
iduronate-2-sulfatase, with irradiation.
Inventors: |
Mann, David M.;
(Gaithersburg, MD) ; Burgess, Wilson; (Clifton,
VA) ; Drohan, William N.; (Springfield, VA) ;
Griko, Yuri; (Gaithersburg, MD) ; MacPhee, Martin
J.; (Montgomery Village, MD) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Clearant Inc.
|
Family ID: |
25478847 |
Appl. No.: |
10/840425 |
Filed: |
May 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10840425 |
May 7, 2004 |
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09942938 |
Aug 31, 2001 |
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6749851 |
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Current U.S.
Class: |
422/22 |
Current CPC
Class: |
A61P 1/12 20180101; A61P
13/12 20180101; A61P 19/02 20180101; A61P 3/00 20180101; A61P 1/18
20180101; A61P 25/28 20180101; A61P 19/00 20180101; A61P 1/06
20180101; A61P 21/00 20180101; A61P 7/06 20180101; A61L 2/007
20130101; A61L 2/0082 20130101; A61L 2/0029 20130101; A61P 27/16
20180101; A61L 2/0041 20130101; A61L 2/0052 20130101; A61P 25/04
20180101; A61P 17/00 20180101; A61P 7/00 20180101; A61P 3/08
20180101; A61P 17/16 20180101; A61L 2/0011 20130101; A61P 9/00
20180101; A61P 11/00 20180101; A61P 27/02 20180101; A61P 1/16
20180101; A61L 2/0035 20130101; A61P 1/14 20180101; A61L 2/0047
20130101; C12N 13/00 20130101; A61P 43/00 20180101 |
Class at
Publication: |
422/022 |
International
Class: |
A61L 002/08 |
Claims
1. (cancelled)
2. A method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation, said method comprising: (i)
adding to said preparation of one more or digestive enzymes at
least one stabilizer in an amount effective to protect said
preparation of one or more digestive enzymes from said radiation;
and (ii) irradiating said preparation of one or more digestive
enzymes with a suitable radiation at an effective rate for a time
effective to sterilize said preparation of one or more digestive
enzymes.
3. A method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation, said method comprising: (i)
reducing the residual solvent content of said preparation of one or
more digestive enzymes to a level effective to protect said
preparation of one or more digestive enzymes from said radiation;
and (ii) irradiating said preparation of one or more digestive
enzymes with a suitable radiation at an effective rate for a time
effective to sterilize said preparation of one or more digestive
enzymes.
4. A method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation, said method comprising: (i)
reducing the temperature of said preparation of one or more
digestive enzymes to a level effective to protect said preparation
of one or more digestive enzymes from said radiation; and (ii)
irradiating said preparation of one or more digestive enzymes with
a suitable radiation at an effective rate for a time effective to
sterilize said preparation of one or more digestive enzymes.
5-85. (cancelled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for sterilizing
preparations of digestive enzymes to reduce the level of one or
more active biological contaminants or pathogens therein, such as
viruses, bacteria (including inter- and intracellular bacteria,
such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia,
rickettsias), yeasts, molds, fungi, prions or similar agents
responsible, alone or in combination, for TSEs and/or single or
multicellular parasites. The present invention particularly relates
to methods of sterilizing preparations of digestive enzymes, such
as trypsin, .alpha.-galactosidase and iduronate 2-sulfatase, with
irradiation.
BACKGROUND OF THE INVENTION
[0002] The principal foods upon which an organism, such as a human,
survives can be broadly categorized as carbohydrates, fats and
proteins. These substances, however, are useless as nutrients
without the process of digestion to break down foods.
[0003] Digestion of carbohydrates begins in the mouth and stomach.
Saliva contains the enzyme ptyalin (an alpha-amylase), which
hydrolyses starch into maltose and other small polymers of glucose.
The pancreatic alpha-amylase is similar to the salivary ptyalin,
but several times as powerful. Therefore, soon after chyme empties
into the duodenum and mixes with pancreatic juice, virtually all of
the starches are converted into disaccharides and small glucose
polymers. These disaccharides and small glucose polymers are
hydrolysed into monosaccharides by intestinal epithelial
enzymes.
[0004] Digestion of proteins begins in the stomach. The enzyme
pepsin, which is produced in the stomach, digests collagen, a major
constituent of the intercellular connective tissue of meats. This
enzymatic reaction is essential so that other digestive enzymes can
penetrate meats and digest the cellular proteins. Consequently, in
people who lack peptic activity in the stomach, the ingested meats
are not well penetrated by these other digestive enzymes and so are
poorly absorbed.
[0005] Most protein digestion results from the actions of the
pancreatic proteolytic enzymes. Proteins leaving the stomach in the
form of proteoses, peptones and large polypeptides are digested
into dipeptides, tripeptides and the like by pancreatic proteolytic
enzymes or polypeptidases. Trypsin and chymotrypsin split protein
molecules into smaller polypeptides at specific peptide linkages,
while carboxypolypeptidase cleaves amino acids from the carboxyl
ends of polypeptides. Proelastase gives rise to elastase, which in
turn digests the elastin fibers that hold together most meat.
[0006] Further digestion of polypeptides takes place in the
intestinal lumen.
[0007] Aminopolypeptidase and several polypeptidases split large
polypeptides into dipeptides, tripeptides and amino acids, which
are transported into the enterocytes that line the intestinal
villi. Inside the enterocytes, other polypeptidases split the
remaining peptides into their constituent amino acids, which then
enter the blood.
[0008] Digestion of fats first requires emulsification by bile
acids and lecithin, which increase the surface area of the fats up
to 1000-fold. Because lipases are water-soluble digestive enzymes
that can bind only on the surface of a fat globule, this
emulsification process is important for the complete digestion of
fat. The most important digestive enzyme in the digestion of
triglycerides is pancreatic lipase, which breaks these down into
free fatty acids and 2-monoglycerides. After these free fatty acids
and monoglycerides enter the enterocytes, they are generally
recombined into new triglyerides. A few monoglycerides, however,
are further digested by intracellular lipases into free fatty
acids.
[0009] Digestion therefore continues after the breakdown and uptake
of nutrients into the various cells of the body. Intracellular
enzymes, such as intracellular lipases, are involved in the uptake,
breakdown, transport, storage, release, metabolism and catabolism
of nutrients into forms required and useable by the cell(s) of an
organism at various places and times. This includes storage of
lipids and their metabolism into energy sources as well as their
catabolism and synthesis into other useful compounds. Digestion may
also occur as a part of an organism's normal process(es) of tissue
generation and regeneration or repair of degraded, damaged or
abnormal tissue(s) or molecules. It may also be a feature of or
result from apoptosis, immune reactions, infections, neoplasms and
other abnormal or disease states of an organism.
[0010] Preparations of digestive enzymes are therefore often
provided therapeutically to humans and animals.
[0011] For example, in cases of pancreatitis and lack of pancreatic
secretion, preparations of certain pancreatic enzymes, including
combinations of lipase, protease and amylase (such as Creon.TM.,
Cotazym.TM., Donnazyme.TM., Ku-Zyme.TM. HP, Pancrease.TM. and
Pancrease.TM. MT, Ultrase.TM. and Ultrase.TM. MT, Viokase.TM., and
Zymase.TM.) and combinations of lipase, protease, amylase and
cellulase (such as Ku-Zyme.TM. and Kutrase.TM.), are administered
to ensure proper patient nutrition. The digestive enzymes of
particular interest, for example in replacement therapy in humans
and animals, therefore include pancreatic digestive enzymes, such
as trypsin and chymotrypsin, and functional mutants, variants and
derivatives thereof.
[0012] Trypsin is an enzyme that acts to degrade protein; it is
often referred to as a digestive enzyme, or proteinase. In the
digestive process, trypsin acts with the other proteinases to break
down dietary protein molecules to their component peptides and
amino acids. Trypsin continues the process of digestion (begun in
the stomach) in the small intestine where a slightly alkaline
environment (about pH 8) promotes its maximal enzymatic activity.
Trypsin, produced in an inactive form by the pancreas, is
remarkably similar in chemical composition and in structure to the
other chief pancreatic proteinase, chymotrypsin. Both enzymes also
appear to have similar mechanisms of action; residues of histidine
and serine are found in the active sites of both. The chief
difference between the two molecules seems to be in their
specificity, that is, each is active only against the peptide bonds
in protein molecules that have carboxyl groups donated by certain
amino acids. For trypsin these amino acids are arginine and lysine,
for chymotrypsin they are tyrosine, phenylalanine, tryptophan,
methionine, and leucine. Trypsin is the most discriminating of all
the digestive enzymes in terms of the restricted number of chemical
bonds that it will attack.
[0013] Preparations of other digestive enzymes, such as
glycosidases, are likewise administered therapeutically to human
patients. For example, Fabry disease is an X-linked recessive
glycolipid storage disorder caused by a deficiency of the lysosomal
enzyme .alpha.-galactosidase A. Clinical manifestations of Fabry
disease included recurrent episodes severe pain and progressive
renal, cardiac and cerebrovascular deterioration with death usually
occurring in the fourth to sixth decade of life. Enzyme replacement
therapy by infusion of a preparation of .alpha.-galactosidase A has
been tested and found to be a promising potential therapy for this
condition (Schiffmann, et al., "Enzyme Replacement Therapy in Fabry
Disease: A Randomized Controlled Trial." JAMA, Jun. 6, 2001, Vol.
285, No. 21, pp. 2743-2749.). Glycogen Storage Disease Type II
(also known as Acid Maltase Deficiency or Pompe Disease) is another
genetically transmitted storage disorder. In GSD-II, the patient
suffers from a deficiency of acid maltase enzyme, which breaks down
glycogen in muscle cells. Clinical manifestations of GSD-II include
progressive muscle weakness due to a build up of glycogen in muscle
tissues, eventually resulting in respiratory and/or cardiac
failure. Preparations of glycosidases, or functional mutants or
variants or derivatives thereof, are therefore also of particular
interest for therapeutic use.
[0014] Niemann-Pick Disease is also a genetically transmitted
metabolic disorder in which harmful quantities of a fatty
substance, sphingomyelin, accumulate in the spleen, liver, lungs,
bone marrow and brain. Patients suffer from a deficiency of
sphingomyelinases, which initiates the biodegradation of
sphinogmyelin. Clinical manifestations include enlargement of the
spleen and liver, and frequently results in death, particularly for
pediatric patients.
[0015] Gaucher's Disease is a somewhat-similar genetically
transmitted disorder, in which harmful quantities of another fatty
substance, glucocerebroside, accumulate in the spleen, liver,
lungs, bone marrow and brain. Patients suffer from a deficiency in
.beta.-glucocerebrosidase, which catalyzes the first step in the
biodegradation of glucocerebroside, which arises from the
biodegradation of old red and white blood cells. Clinical
manifestations include enlargement of the spleen and liver, low
blood platelets, fatigue and, in certain forms, progressive brain
damage. Enzyme replacement therapy by infusion of a preparation of
a modified form of glucocerebrosidase, known as algucerase
(Ceredase.TM.) has been tested and found to be a promising
potential therapy for this condition (Barton, et al., "Replacement
Therapy for Enzyme Deficiency: Macrophage-targeted
Glucocerebrosidase for Gaucher's Disease." New Engl. J. Med., May
23, 1991.).
[0016] Mucopolysaccharidoses are a group of inherited metabolic
disorders caused by a deficiency in the lysosomal enzymes needed to
break down mucopolysaccharides, long chains of sugar molecules used
to build connective tissue and organs in the body. A deficiency in
one or more of these enzymes cases a build up of excess amount in
the body, causing progressive damage and eventual death. Among
these disorders are Hurler, Scheie and Hurler/Scheie syndromes (the
most severe form, occurs in infancy with death resulting before age
10 years, symptoms include clouding of the cornea and progressive
physical and mental disability, caused by a deficiency in
.alpha.-L-iduronidase), Hunter syndrome (affects juveniles with
death usually resulting by age 15 years, symptoms include joint
stiffness, mental deterioration, dwarfing and progressive deafness,
caused by a deficiency in iduronate-2-sulfatase), Sanfillipo
syndrome (death usually occurs by late teens, symptoms include
progressive dementia and mental deterioration in childhood, caused
by a deficiency in heparan N-sulfatase,
.alpha.-N-acetylglucosaminadase, acetyl-CoA-glucosaminide
acetyltransferase and/or N-acetylglucosamine-6-s- ulfatase),
Morquio syndrome (appears in infancy, symptoms include severe
dwarfing and corneal clouding, cardiac or respiratory disease may
cause death in third or fourth decade of like, caused by a
deficiency in galactosamine-6-sulfatase and/or
.beta.-galactosidase), Maroteauz-Lamy syndrome (resembles Hurler
syndrome, onset in infancy, but no mental disability, death usually
occurs in second or third decade of life, caused by a deficiency in
arylsulfatase B), and Sly disease (symptoms include corneal
clouding, skeletal irregularities, and enlargement of the liver and
spleen, caused by a deficiency in .beta.-glucuronidase). Hunter
syndrome is particularly linked to a deficiency in
iduronate-2-sulfatase, which catalyzes the breakdown of heparan
sulfate and dermatan sulfate, and it has been suggested that this
condition can be treated by administration of variant forms of the
enzyme (U.S. Pat. No. 6,153,188). The digestive of particular
interest, for example in therapy in humans and animals, therefore
also include iduronate-2-sulfatase and functional mutants, variants
and derivatives thereof.
[0017] Multiple Sulfatase Deficiency (also known as Disorder of
Confication 13 or Mucosulfatidosis) is another hereditary metabolic
disorder characterized by impairment of all known sulfatase enzymes
(including arylsulfatases A, B and C, two steroid sulfatases and
four other sulfatases). Clinical manifestations include coarse
facial features, deafness, an enlarged liver and spleen,
abnormalities of the skeleton (including lumbar kyphosis) and dry,
scaly skin (ichthyosis).
[0018] Similarly, preparations of digestive enzymes are
administered to humans and animals to improve nutrition.
[0019] For example, in cases of lactose intolerance, preparations
of lactase (such as Lactaid.TM.) are administered to humans in need
thereof. Lactose intolerance is characterized by gastrointestinal
discomfort, including gas, bloating, crampls and diarrhea, after
the consumption of milk or milk-containing products. The digestive
enzymes of particular interest, for example in therapy in humans
and animals, therefore also include lactase and functional mutants,
variants and derivatives thereof.
[0020] Likewise, preparations of galactosidases (such as Beano.TM.
or Nutritek.TM. Alpha Galactosidase) are administered to humans in
need thereof. Such products improve digestion of sugars found in
foods including legumes and cruciferous vegetables and reduce
effects generally associated with the foods, such as gas and
bloating.
[0021] Preparations of digestive enzymes that are prepared for
human, veterinary, diagnostic and/or experimental use may contain
unwanted and potentially dangerous biological contaminants or
pathogens, such as viruses, bacteria (including inter- and
intracellular bacteria, such as mycoplasmas, ureaplasmas,
nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions
or similar agents responsible, alone or in combination, for TSEs
and/or single or multicellular parasites. Consequently, it is of
utmost importance that any biological contaminant in the
preparation be inactivated before the product is used. This is
especially critical when the preparation is to be administered
directly to a patient, for example in human therapy corrected or
treated by intravenous, intramuscular or other forms of injection.
This is also critical for the various preparations that are
prepared in media or via culture of cells or recombinant cells
which contain various types of plasma and/or plasma derivatives or
other biological materials or are used to prepare biological
materials for human use and which may be subject to mycoplasma,
prion, bacterial, viral and/or other biological contaminants or
pathogens.
[0022] Most procedures for producing preparations of digestive
enzymes have involved methods that screen or test the preparation
for one or more particular biological contaminants or pathogens
rather than removal or inactivation of the contaminant(s) and/or
pathogen(s) from the preparation. Preparations that test positive
for a biological contaminant or pathogen are merely not used.
Examples of screening procedures include the testing for a
particular virus in human blood from blood donors. Such procedures,
however, are not always reliable and are not able to detect the
presence of certain viruses, particularly in very low numbers, and
in the case of as yet unknown viruses or other contaminants or
pathogens that may be in blood. This reduces the value or certainty
of the test in view of the consequences associated with a false
negative result. False negative results can be life threatening in
certain cases, for example in the case of Acquired Immune
Deficiency Syndrome (AIDS). Furthermore, in some instances it can
take weeks, if not months, to determine whether or not the
preparation is contaminated. Therefore, it would be desirable to
apply techniques that would kill or inactivate biological
contaminants and pathogens during and/or after manufacturing the
preparation of digestive enzymes.
[0023] In conducting experiments to determine the ability of
technologies to inactivate viruses, the actual viruses of concern
are seldom utilized. This is a result of safety concerns for the
workers conducting the tests, and the difficulty and expense
associated with the containment facilities and waste disposal. In
their place, model viruses of the same family and class are
used.
[0024] In general, it is acknowledged that the most difficult
viruses to inactivate are those with an outer shell made up of
proteins, and that among these, the most difficult to inactivate
are those of the smallest size. This has been shown to be true for
gamma irradiation and most other forms of radiation as these
viruses' diminutive size is associated with a small genome. The
magnitude of direct effects of radiation upon a molecule are
directly proportional to the size of the molecule, that is the
larger the target molecule, the greater the effect. As a corollary,
it has been shown for gamma-irradiation that the smaller the viral
genome, the higher the radiation dose required to inactive it.
[0025] Among the viruses of concern for both human and
animal-derived preparations, the smallest, and thus most difficult
to inactivate, belong to the family of Parvoviruses and the
slightly larger protein-coated Hepatitis virus. In humans, the
Parvovirus B19, and Hepatitis A are the agents of concern. In
porcine-derived materials, the smallest corresponding virus is
Porcine Parvovirus. Since this virus is harmless to humans, it is
frequently chosen as a model virus for the human B19 Parvovirus.
The demonstration of inactivation of this model parvovirus is
considered adequate proof that the method employed will kill human
B19 virus and Hepatitis A, and by extension, that it will also kill
the larger and less hardy viruses such as HIV, CMV, Hepatitis B and
C and others.
[0026] More recent efforts have focussed on methods to remove or
inactivate contaminants in the products. Such methods include heat
treating, filtration and the addition of chemical inactivants or
sensitizers to the product.
[0027] Heat treatment requires that the product be heated to
approximately 60.degree. C. for about 70 hours which can be
damaging to sensitive products. In some instances, heat
inactivation can actually destroy 50% or more of the biological
activity of the product.
[0028] Filtration involves filtering the product in order to
physically remove contaminants. Unfortunately, this method may also
remove products that have a high molecular weight. Further, in
certain cases, small viruses and similarly sized contaminants and
pathogens, such as prions, may not be removed by the filter.
[0029] The procedure of chemical sensitization involves the
addition of noxious agents which bind to the DNA/RNA of the virus
and which are activated either by UV or other radiation. This
radiation produces reactive intermediates and/or free radicals
which bind to the DNA/RNA of the virus, break the chemical bonds in
the backbone of the DNA/RNA, and/or cross-link or complex it in
such a way that the virus can no longer replicate. This procedure
requires that unbound sensitizer is washed from products since the
sensitizers are toxic, if not mutagenic or carcinogenic, and cannot
be administered to a patient.
[0030] Irradiating a product with gamma radiation is another method
of sterilizing a product. Gamma radiation is effective in
destroying viruses and bacteria when given in high total doses
(Keathly et al., "Is There Life After Irradiation? Part 2,"
BioPharm July-August, 1993, and Leitman, Use of Blood Cell
Irradiation in the Prevention of Post Transfusion Graft-vs-Host
Disease," Transfusion Science 10:219-239 (1989)). The published
literature in this area, however, teaches that gamma radiation can
be damaging to radiation sensitive products, such as blood, blood
products, enzymes, protein and protein-containing products. In
particular, it has been shown that high radiation doses are
injurious to red cells, platelets and granulocytes (Leitman). U.S.
Pat. No. 4,620,908 discloses that protein products must be frozen
prior to irradiation in order to maintain the viability of the
protein product. This patent concludes that "[i]f the gamma
irradiation were applied while the protein material was at, for
example, ambient temperature, the material would be also completely
destroyed, that is the activity of the material would be rendered
so low as to be virtually ineffective". Unfortunately, many
sensitive biological materials, such as monoclonal antibodies
(Mab), may lose viability and activity if subjected to freezing for
irradiation purposes and then thawing prior to administration to a
patient.
[0031] In view of the difficulties discussed above, there remains a
need for methods of sterilizing preparations of one or more
digestive enzymes that are effective for reducing the level of
active biological contaminants or pathogens without an adverse
effect on the preparation.
SUMMARY OF THE INVENTION
[0032] Accordingly, it is an object of the present invention to
provide methods of sterilizing preparations of digestive enzymes by
reducing the level of active biological contaminants or pathogens
without adversely effecting the preparation. Other objects,
features and advantages of the present invention will be set forth
in the detailed description of preferred embodiments that follows,
and in part will be apparent from the description or may be learned
by practice of the invention. These objects and advantages of the
invention will be realized and attained by the compositions and
methods particularly pointed out in the written description and
claims hereof.
[0033] In accordance with these and other objects, a first
embodiment of the present invention is directed to a method for
sterilizing a preparation of one or more digestive enzymes that is
sensitive to radiation comprising irradiating the preparation of
one or more digestive enzymes with radiation for a time effective
to sterilize the material at a rate effective to sterilize the
material and to protect the material from radiation.
[0034] Another embodiment of the present invention is directed to a
method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation comprising: (i) adding to a
preparation of one or more digestive enzymes at least one
stabilizer in an amount effective to protect the preparation of one
or more digestive enzymes from radiation; and (ii) irradiating the
preparation of one or more digestive enzymes with radiation at an
effective rate for a time effective to sterilize the material.
[0035] Another embodiment of the present invention is directed to a
method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation comprising: (i) reducing the
residual solvent content of a preparation of one or more digestive
enzymes to a level effective to protect the preparation of one or
more digestive enzymes from radiation; and (ii) irradiating the
preparation of one or more digestive enzymes with radiation at an
effective rate for a time effective to sterilize the preparation of
one or more digestive enzymes.
[0036] Another embodiment of the present invention is directed to a
method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation comprising: (i) reducing the
temperature of a preparation of one or more digestive enzymes to a
level effective to protect the preparation of one or more digestive
enzymes from radiation; and (ii) irradiating the preparation of one
or more digestive enzymes with radiation at an effective rate for a
time effective to sterilize the preparation of one or more
digestive enzymes.
[0037] Another embodiment of the present invention is directed to a
method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation comprising: (i) applying to
the preparation of one or more digestive enzymes a stabilizing
process selected from the group consisting of: (a) reducing the
residual solvent content of a preparation of one or more digestive
enzymes, (b) adding to the preparation of one or more digestive
enzymes at least one stabilizer, and (c) reducing the temperature
of the preparation of one or more digestive enzymes; and (ii)
irradiating the preparation of one or more digestive enzymes with
radiation at an effective rate for a time effective to sterilize
the preparation of one or more digestive enzymes, wherein the
stabilizing process and the rate of irradiation are together
effective to protect the preparation of one or more digestive
enzymes from radiation.
[0038] Another embodiment of the present invention is directed to a
method for sterilizing a preparation of one or more digestive
enzymes that is sensitive to radiation comprising: (i) applying to
the preparation of one or more digestive enzymes at least two
stabilizing processes selected from the group consisting of: (a)
reducing the residual solvent content of a preparation of one or
more digestive enzymes, (b) adding to the preparation of one or
more digestive enzymes at least one stabilizer, and (c) reducing
the temperature of the preparation of one or more digestive
enzymes; and (ii) irradiating the preparation of one or more
digestive enzymes with radiation at an effective rate for a time
effective to sterilize the preparation of one or more digestive
enzymes, wherein the stabilizing processes may be performed in any
order and are together effective to protect the preparation of one
or more digestive enzymes from radiation.
[0039] The invention also provides a biological composition
comprising at least one preparation of one or more digestive
enzymes and a least one stabilizer in an amount effective to
preserve the preparation of one or more digestive enzymes for its
intended use following sterilization with radiation.
[0040] The invention also provides a biological composition
comprising at least one preparation of one or more digestive
enzymes in which the residual solvent content has been reduced to a
level effective to preserve the preparation of one or more
digestive enzymes for its intended use following sterilization with
radiation.
[0041] The invention also provides a biological composition
comprising at least one preparation of one or more digestive
enzymes and at least one stabilizer in which the residual solvent
content has been reduced and wherein the amount of stabilizer and
level of residual solvent content are together effective to
preserve the preparation of one or more digestive enzymes for its
intended use following sterilization with radiation.
[0042] The invention also provides a biological composition
comprising at least one preparation of one or more digestive
enzymes wherein the total protein concentration of the preparation
is effective to preserve the preparation of one or more digestive
enzymes for its intended use following sterilization with
radiation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIGS. 1A-1B are graphs showing the activity of lyophilized
trypsin following gamma irradiation in the absence or presence of a
stabilizer and at varying levels of residual solvent content.
[0044] FIG. 2 is a graph showing the activity of liquid or
lyophilized trypsin following gamma irradiation in the presence of
a stabilizer and at varying pH levels.
[0045] FIGS. 3A-3B are graphs showing the activity of lyophilized
trypsin following gamma irradiation in the absence or presence of a
stabilizer.
[0046] FIGS. 4A-4B are graphs showing the activity of lyophilized
trypsin following gamma irradiation in the absence or presence of a
stabilizer and at varying levels of residual solvent content.
[0047] FIGS. 5A-5B are graphs showing the activity of lyophilized
trypsin following gamma irradiation in the absence or presence of a
stabilizer and at varying levels of residual solvent content.
[0048] FIG. 6 is a graph showing the activity of trypsin suspended
in polypropylene glycol following gamma irradiation at varying
levels of residual solvent content.
[0049] FIG. 7 is a graph showing the activity of trypsin following
gamma irradiation in an aqueous solution at varying concentrations
of stabilizers.
[0050] FIGS. 8A-8B are gels showing the protective effect of
ascorbate (200 mM) and a combination of ascorbate (200 mM) and
Gly-Gly (200 mM) on two different frozen enzyme preparations (a
glycosidase and a sulfatase).
[0051] FIG. 9 is a graph showing the protective effect of
stabilizers on a frozen glycosidase preparation.
[0052] FIG. 10 shows the protective effect of ascorbate on two
different lyophilized enzyme preparations (a glycosidase and a
sulfatase).
[0053] FIGS. 11A-11C are gels showing the protective effect of
ascorbate (200 mM) and a combination of ascorbate (200 mM) and
Gly-Gly (200 mM) on a lyophilized glycosidase preparation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] A. Definitions
[0055] Unless defined otherwise, all technical and scientific terms
used herein are intended to have the same meaning as is commonly
understood by one of ordinary skill in the relevant art.
[0056] As used herein, the singular forms "a," "an," and "the"
include the plural reference unless the context clearly dictates
otherwise.
[0057] As used herein, the term "preparation of one or more
digestive enzymes" is intended to mean any preparation derived or
obtained from a living organism that contains one or more of
enzymes involved in the breakdown or conversion of one substance
into a second substance, particularly protein(s), lipid(s) and/or
cabohydrate(s). Illustrative examples of digestive enzymes include,
but are not limited to, intracellular and intercellular enzymes
produced by, present in or introduced into the digestive tract of
any living organism, or involved in the metabolism, catabolism,
storage and mobilization of externally or internally-derived
nutrients or the breakdown products of tissue and/or cellular
repair, regeneration, or removal, such as the following: pancreatic
enzymes, including pancreatic proteolytic enzymes, such as trypsin
and chymotrypsin, pancreatic lipase and pancreatic amylase;
salivary enzymes, such as ptyalin; intestinal enzymes, including
intestinal polypeptidases, intestinal amylases and intestinal
lipases; glycosidases, such as .alpha.-galactosidase; and
sulfatases, such as iduronodate-2-sulfatase.
[0058] As used herein, the term "sterilize" is intended to mean a
reduction in the level of at least one active biological
contaminant or pathogen found in the preparation being treated
according to the present invention.
[0059] As used herein, the term "biological contaminant or
pathogen" is intended to mean a contaminant or pathogen that, upon
direct or indirect contact with a preparation of one or more
digestive enzymes, may have a deleterious effect on the digestive
enzymes or upon a recipient thereof. Such biological contaminants
or pathogens include the various viruses, bacteria (including
inter- and intracellular bacteria, such as mycoplasmas,
ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds,
fungi, prions or similar agents responsible, alone or in
combination, for TSEs and/or single or multicellular parasites
known to those of skill in the art to generally be found in or
infect preparations of digestive enzymes. Examples of biological
contaminants or pathogens include, but are not limited to, the
following: viruses, such as human immunodeficiency viruses and
other retroviruses, herpes viruses, filliviruses, circoviruses,
paramyxoviruses, cytomegaloviruses, hepatitis viruses (including
hepatitis A, B and C), pox viruses, toga viruses, Epstein-Barr
viruses and parvoviruses; bacteria, such as Escherichia, Bacillus,
Campylobacter, Streptococcus and Staphalococcus; nanobacteria;
parasites, such as Trypanosoma and malarial parasites, including
Plasmodium species; yeasts; molds; mycoplasmas and ureaplasmas;
chlamydia; rickettsias, such as Coxiella burnetti; and prions and
similar agents responsible, alone or in combination, for one or
more of the disease states known as transmissible spongiform
encephalopathies (TSEs) in mammals, such as scrapie, transmissible
mink encephalopathy, chronic wasting disease (generally observed in
mule deer and elk), feline spongiform encephalopathy, bovine
spongiform encephalopathy (mad cow disease); Creutzfeld-Jakob
disease (including variant or new variant CJD), Fatal Familial
Insomnia; Gerstmann-Straeussler-Scheinker syndrome; kuru; and
Alpers syndrome. As used herein, the term "active biological
contaminant or pathogen" is intended to mean a biological
contaminant or pathogen that is capable of causing a deleterious
effect, either alone or in combination with another factor, such as
a second biological contaminant or pathogen or a native protein
(wild-type or mutant) or antibody, in the preparation of digestive
enzymes and/or a recipient thereof.
[0060] As used herein, the term "a biologically compatible
solution" is intended to mean a solution to which a preparation of
one or more digestive enzymes may be exposed, such as by being
suspended or dissolved therein, and remain viable, i.e., retain its
essential biological and physiological characteristics.
[0061] As used herein, the term "a biologically compatible buffered
solution" is intended to mean a biologically compatible solution
having a pH and osmotic properties (e.g., tonicity, osmolality
and/or oncotic pressure) suitable for maintaining the integrity of
the material(s) therein. Suitable biologically compatible buffered
solutions typically have a pH between 4 and 8.5 and are isotonic or
only moderately hypotonic or hypertonic. Biologically compatible
buffered solutions are known and readily available to those of
skill in the art.
[0062] As used herein, the term "stabilizer" is intended to mean a
compound or material that reduces damage to the biological material
being irradiated to a level that is insufficient to preclude the
safe and effective use of the material. Illustrative examples of
stabilizers include, but are not limited to, the following:
antioxidants; free radical scavengers, including spin traps;
combination stabilizers, i.e. stabilizers which are effective at
quenching both Type I and Type II photodynamic reactions; and
ligands, such as heparin, that stabilize the molecules to which
they bind. Preferred examples of stabilizers include, but are not
limited to, the following: ethanol; acetone; fatty acids, including
6,8-dimercapto-octanoic acid (lipoic acid) and its derivatives and
analogues (alpha, beta, dihydro, bisno and tetranor lipoic acid),
thioctic acid, 6,8-dimercapto-octanoic acid, dihydrolopoate
(DL-6,8-dithioloctanoic acid methyl ester), lipoamide, bisonor
methyl ester and tatranor-dihydrolipoic acid, furan fatty acids,
cleic and linoleic and palmitic acids and their salts and
derivatives; flavonoids, phenylpropaniods, and flavenols, such as
quercetin, rutin and its derivatives, apigenin, aminoflavone,
catechin, hesperidin and, naringin; carotenes, including
beta-carotene; Co-Q10; xanthophylls; polyhydric alcohols, such as
glycerol, mannitol; sugars, such as xylose, glucose, ribose,
mannose, fructose and trehalose; amino acids and derivatives
thereof, such as histidine, N-acetylcysteine (NAC), glutamic acid,
tryptophan, sodium caprylate, N-acetyl tryptophan and methionine;
azides, such as sodium azide; enzymes, such as Superoxide Dismutase
(SOD) and Catalase; uric acid and its derivatives, such as
1,3-dimethyluric acid and dimethylthiourea; allopurinol; thiols,
such as glutathione and reduced glutathione and cysteine; trace
elements, such as selenium; vitamins, such as vitamin A, vitamin C
(including its derivatives and salts such as sodium ascorbate and
palmitoyl ascorbic acid) and vitamin E (and its derivatives and
salts such as tocopherol acetate and alpha-tocotrienol);
chromanol-alpha-C6; 6-hydroxy-2,5,7,8-tetramethylchro- ma-2
carboxylic acid (Trolox) and derivatives; extraneous proteins, such
as gelatin and albumin; tris-3-methyl-1-phenyl-2-pyrazolin-5-one
(MCI-186); citiolone; puercetin; chrysin; dimethyl sulfoxide
(DMSO); piperazine diethanesulfonic acid (PIPES); imidazole;
methoxypsoralen (MOPS); 1,2-dithiane-4,5-diol; reducing substances,
such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene
(BHT); cholesterol; probucol; indole derivatives; thimerosal;
lazaroid and tirilazad mesylate; proanthenols; proanthocyanidins;
ammonium sulfate; Pegorgotein (PEG-SOD);
N-tert-butyl-alpha-phenylnitrone (PBN);
4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol); mixtures of
ascorbate, urate and Trolox C (Asc/urate/Trolox C); proteins and
peptides, such as glycylglycine and carnosine, in which each amino
acid may be in its D or L form; diosrnin; pupurogalin; gallic acid
and its derivatives including but not limited to propyl gallate,
sodium formaldehyde sulfoxylate and silymarin. Particularly
preferred examples include single stabilizers or combinations of
stabilizers that are effective at quenching both Type I and Type II
photodynamic reactions and volatile stabilizers, which can be
applied as a gas and/or easily removed by evaporation, low pressure
and similar methods.
[0063] As used herein, the term "residual solvent content" is
intended to mean the amount or proportion of freely available
liquid in the preparation of one or more digestive enzymes. Freely
available liquid means the liquid, such as water or an organic
solvent (e.g. ethanol, isopropanol, acetone, polyethylene glycol,
etc.), present in the preparation being sterilized that is not
bound to or complexed with one or more of the non-liquid components
of the preparation. Freely available liquid includes intracellular
water. The residual solvent contents related as water referenced
herein refer to levels determined by the FDA approved, modified
Karl Fischer method (Meyer and Boyd, Analytical Chem., 31:215-219,
1959; May, et al., J. Biol. Standardization, 10:249-259, 1982;
Centers for Biologics Evaluation and Research, FDA, Docket No.
89D-0140, 83-93; 1990) and/or by near infrared spectroscopy.
Quantitation of the residual levels of other solvents may be
determined by means well known in the art, depending upon which
solvent is employed. The proportion of residual solvent to solute
may also be considered to be a reflection of the concentration of
the solute within the solvent. When so expressed, the greater the
concentration of the solute, the lower the amount of residual
solvent.
[0064] As used herein, the term "sensitizer" is intended to mean a
substance that selectively targets viral, bacterial, nanobacterial,
mold, yeast, fungal, prion and/or parasitic contaminants or
pathogens, rendering them more sensitive to inactivation by
radiation, therefore permitting the use of a lower rate or dose of
radiation and/or a shorter time of irradiation than in the absence
of the sensitizer. Illustrative examples of suitable sensitizers
include, but are not limited to, the following: psoralen and its
derivatives and analogs (including 3-carboethoxy psoralens);
inactines and their derivatives and analogs; angelicins, khellins
and coumarins which contain a halogen substituent and a water
solubilization moiety, such as quaternary ammonium ion or
phosphonium ion; nucleic acid binding compounds; brominated
hematoporphyrin; phthalocyanines; purpurins; porphorins;
halogenated or metal atom-substituted derivatives of
dihematoporphyrin esters, hematoporphyrin derivatives,
benzoporphyrin derivatives, hydrodibenzoporphyrin dimaleimade,
hydrodibenzoporphyrin, dicyano disulfone, tetracarbethoxy
hydrodibenzoporphyrin, and tetracarbethoxy hydrodibenzoporphyrin
dipropionamide; doxorubicin and daunomycin, which may be modified
with halogens or metal atoms; netropsin; BD peptide, S2 peptide;
S-303 (ALE compound); dyes, such as hypericin, methylene blue,
eosin, fluoresceins (and their derivatives), flavins, merocyanine
540; photoactive compounds, such as bergapten; and SE peptide.
[0065] As used herein, the term "radiation" is intended to mean
radiation of sufficient energy to sterilize at least some component
of the irradiated preparation of one or more digestive enzymes.
Types of radiation include, but are not limited to, the following:
(i) corpuscular (streams of subatomic particles such as neutrons,
electrons, and/or protons); and (ii) electromagnetic (originating
in a varying electromagnetic field, such as radio waves, visible
(both mono and polychromatic) and invisible light, infrared,
ultraviolet radiation, x-radiation, and gamma rays and mixtures
thereof). Such radiation is often described as either ionizing
(capable of producing ions in irradiated materials) radiation, such
as gamma rays, and non-ionizing radiation, such as visible light.
The sources of such radiation may vary and, in general, the
selection of a specific source of radiation is not critical
provided that sufficient radiation is given in an appropriate time
and at an appropriate rate to effect sterilization. In practice,
gamma radiation is usually produced by isotopes of Cobalt or
Cesium, while X-rays are produced by machines that emit
X-radiation, and electrons are often used to sterilize materials in
a method known as "E-beam" irradiation that involves their
production via a machine.
[0066] As used herein, the term "to protect" is intended to mean to
reduce any damage to the preparation of one or more digestive
enzymes being irradiated, that would otherwise result from the
irradiation of that material, to a level that is insufficient to
preclude the safe and effective use of the material following
irradiation. In other words, a substance or process "protects" a
preparation of one or more digestive enzymes from radiation if the
presence of that substance or carrying out that process results in
less damage to the material from irradiation than in the absence of
that substance or process. Thus, a preparation of one or more
digestive enzymes may be used safely and effectively after
irradiation in the presence of a substance or following performance
of a process that "protects" the material, but could not be used
safely and effectively after irradiation under identical conditions
but in the absence of that substance or the performance of that
process.
[0067] B. Particularly Preferred Embodiments
[0068] A first preferred embodiment of the present invention is
directed to a method for sterilizing a preparation of one or more
digestive enzymes that is sensitive to radiation comprising
irradiating the preparation of one or more digestive enzymes with
radiation for a time effective to sterilize the material at a rate
effective to sterilize the material and to protect the material
from radiation.
[0069] Another preferred embodiment of the present invention is
directed to a method for sterilizing a preparation of one or more
digestive enzymes that is sensitive to radiation comprising: (i)
adding to a preparation of one or more digestive enzymes at least
one stabilizer in an amount effective to protect the preparation of
one or more digestive enzymes from radiation; and (ii) irradiating
the preparation of one or more digestive enzymes with radiation at
an effective rate for a time effective to sterilize the
material.
[0070] Another preferred embodiment of the present invention is
directed to a method for sterilizing a preparation of one or more
digestive enzymes that is sensitive to radiation comprising: (i)
reducing the residual solvent content of a preparation of one or
more digestive enzymes to a level effective to protect the
preparation of one or more digestive enzymes from radiation; and
(ii) irradiating the preparation of one or more digestive enzymes
with radiation at an effective rate for a time effective to
sterilize the preparation of one or more digestive enzymes.
[0071] Another preferred embodiment of the present invention is
directed to a method for sterilizing a preparation of one or more
digestive enzymes that is sensitive to radiation comprising: (i)
reducing the temperature of a preparation of one or more digestive
enzymes to a level effective to protect the preparation of one or
more digestive enzymes from radiation; and (ii) irradiating the
preparation of one or more digestive enzymes with radiation at an
effective rate for a time effective to sterilize the preparation of
one or more digestive enzymes.
[0072] Another preferred embodiment of the present invention is
directed to a method for sterilizing a preparation of one or more
digestive enzymes that is sensitive to radiation comprising: (i)
applying to the preparation of one or more digestive enzymes a
stabilizing process selected from the group consisting of: (a)
reducing the residual solvent content of a preparation of one or
more digestive enzymes, (b) adding to the preparation of one or
more digestive enzymes at least one stabilizer, and (c) reducing
the temperature of the preparation of one or more digestive
enzymes; and (ii) irradiating the preparation of one or more
digestive enzymes with radiation at an effective rate for a time
effective to sterilize the preparation of one or more digestive
enzymes, wherein the stabilizing process and the rate of
irradiation are together effective to protect the preparation of
one or more digestive enzymes from radiation.
[0073] Another preferred embodiment of the present invention is
directed to a method for sterilizing a preparation of one or more
digestive enzymes that is sensitive to radiation comprising: (i)
applying to the preparation of one or more digestive enzymes at
least two stabilizing processes selected from the group consisting
of: (a) reducing the residual solvent content of a preparation of
one or more digestive enzymes, (b) adding to the preparation of one
or more digestive enzymes at least one stabilizer, and (c) reducing
the temperature of the preparation of one or more digestive
enzymes; and (ii) irradiating the preparation of one or more
digestive enzymes with radiation at an effective rate for a time
effective to sterilize the preparation of one or more digestive
enzymes, wherein the stabilizing processes may be performed in any
order and are together effective to protect the preparation of one
or more digestive enzymes from radiation.
[0074] According to certain methods of the present invention, a
stabilizer is added to the preparation of one or more digestive
enzymes prior to irradiation of the preparation of one or more
digestive enzymes with radiation. This stabilizer is added in an
amount that is effective to protect the preparation of one or more
digestive enzymes from the radiation. Suitable amounts of
stabilizer may vary depending upon certain features of the
particular method(s) of the present invention being employed, such
as the nature and characteristics of the particular preparation of
one or more digestive enzymes and/or stabilizer being used, and/or
the intended use of the preparation of one or more digestive
enzymes being irradiated, and can be determined empirically by one
skilled in the art.
[0075] According to certain methods of the present invention, the
residual solvent content of the preparation of one or more
digestive enzymes is reduced prior to irradiation of the
preparation of one or more digestive enzymes with radiation. The
residual solvent content is reduced to a level that is effective to
protect the preparation of one or more digestive enzymes from the
radiation. Suitable levels of residual solvent content may vary
depending upon certain features of the particular method(s) of the
present invention being employed, such as the nature and
characteristics. of the particular preparation of one or more
digestive enzymes and/or stabilizer being used, and/or the intended
use of the preparation of one or more digestive enzymes being
irradiated, and can be determined empirically by one skilled in the
art. There may be preparations for which it is desirable to
maintain the residual solvent content to within a particular range,
rather than a specific value, for example when the solvent, or at
least one of the solvents in a mixture, is also a stabilizer, such
as an alcohol (e.g. ethanol)- or dialkyl ketone (e.g. acetone).
[0076] When the solvent is water, and particularly when the
preparation of one or more digestive enzymes is in a solid phase,
the residual solvent content is generally less than about 15%,
typically less than about 10%, more typically less than about 9%,
even more typically less than about 8%, usually less than about 5%,
preferably less than about 3.0%, more preferably less than about
2.0%, even more preferably less than about 1.0%, still more
preferably less than about 0.5%, still even more preferably less
than about 0.2% and most preferably less than about 0.08%.
[0077] The solvent may preferably be a non-aqueous solvent, more
preferably a non-aqueous solvent that is not prone to the formation
of free-radicals upon irradiation, and most preferably a
non-aqueous solvent that is not prone to the formation of
free-radicals upon irradiation and that has little or no dissolved
oxygen or other gas(es) that is (are) prone to the formation of
free-radicals upon irradiation. Volatile non-aqueous solvents are
particularly preferred, even more particularly preferred are
non-aqueous solvents that are stabilizers, such as ethanol and
acetone.
[0078] In certain embodiments of the present invention, the solvent
may be a mixture of water and a non-aqueous solvent or solvents,
such as ethanol and/or acetone. In such embodiments, the
non-aqueous solvent(s) is preferably a non-aqueous solvent that is
not prone to the formation of free-radicals upon irradiation, and
most preferably a non-aqueous solvent that is not prone to the
formation of free-radicals upon irradiation and that has little or
no dissolved oxygen or other gas(es) that is (are) prone to the
formation of free-radicals upon irradiation. Volatile non-aqueous
solvents are particularly preferred, even more particularly
preferred are non-aqueous solvents that are stabilizers, such as
ethanol and acetone.
[0079] In a preferred embodiment, when the residual solvent is
water, the residual solvent content of a biological material is
reduced by dissolving or suspending the biological material in a
non-aqueous solvent that is capable of dissolving water.
Preferably, such a non-aqueous solvent is not prone to the
formation of free-radicals upon irradiation and has little or no
dissolved oxygen or other gas(es) that is (are) prone to the
formation of free-radicals upon irradiation.
[0080] When the biological material is in a liquid phase, reducing
the residual solvent content may be accomplished by any of a number
of means, such as by increasing the solute concentration. In this
manner, the concentration of the biological material dissolved
within the solvent may be increased to generally at least about
0.5%, typically at least about 1%, usually at least about 5%,
preferably at least about 10%, more preferably at least about 15%,
even more preferably at least about 20%, still even more preferably
at least about 25%, and most preferably at least about 50%.
[0081] In certain embodiments of the present invention, the
residual solvent content of a particular biological material may be
found to lie within a range, rather than at a specific point. Such
a range for the preferred residual solvent content of a particular
biological material may be determined empirically by one skilled in
the art.
[0082] While not wishing to be bound by any theory of operability,
it is believed that the reduction in residual solvent content
reduces the degrees of freedom of the preparation of one or more
digestive enzymes, reduces the number of targets for free radical
generation and may restrict the solubility or diffusion of these
free radicals. Similar results might therefore be achieved by
lowering the temperature of the preparation of one or more
digestive enzymes below its eutectic point or below its freezing
point, or by vitrification to likewise reduce the degrees of
freedom of the preparation of one or more digestive enzymes. These
results may permit the use of a higher rate and/or dose of
radiation than might otherwise be acceptable. Thus, the methods
described herein may be carried out at any temperature that doesn't
result in an unacceptable level of damage to the preparation.
Preferably, the methods described herein are performed at ambient
temperature or below ambient temperature, such as below the
eutectic point or freezing point of the preparation of one or more
digestive enzymes being irradiated.
[0083] In accordance with the methods of the present invention, an
"acceptable level" of damage may vary depending upon certain
features of the particular method(s) of the present invention being
employed, such as the nature and characteristics of the particular
preparation of one or more digestive enzymes and/or stabilizer
being used, and/or the intended use of the preparation of one or
more digestive enzymes being irradiated, and can be determined
empirically by one skilled in the art. An "unacceptable level" of
damage would therefore be a level of damage that would preclude the
safe and effective use of the preparation of one or more digestive
enzymes being sterilized. The particular level of damage in a given
preparation of one or more digestive enzymes may be determined
using any of the methods and techniques known to one skilled in the
art.
[0084] The residual solvent content of a preparation of one or more
digestive enzymes may be reduced by any of the methods and
techniques known to those skilled in the art for reducing solvent
from a preparation of one or more digestive enzymes without
producing an unacceptable level of damage to the preparation. Such
methods include, but are not limited to, evaporation,
concentration, centrifugal concentration, vitrification, addition
of solute, lyophilization (with or without the prior addition of
ascorbate) and spray-drying.
[0085] A particularly preferred method for reducing the residual
solvent content of a preparation of one or more digestive enzymes
is lyophilization, even more preferred is lyophilization following
the addition of ascorbate.
[0086] Another particularly preferred method for reducing the
residual solvent content of a preparation of one or more digestive
enzymes is vitrification, which may be accomplished by any of the
methods and techniques known to those skilled in the art, including
the addition of solute and or additional solutes, such as sucrose,
to raise the eutectic point of the biological material, followed by
a gradual application of reduced pressure to the biological
material in order to remove the residual solvent, such as water.
The resulting glassy material will then have a reduced residual
solvent content.
[0087] According to certain methods of the present invention, the
preparation of one or more digestive enzymes to be sterilized may
be immobilized upon a solid surface by any means known and
available to one skilled in the art. For example, the preparation
of one or more digestive enzymes to be sterilized may be present as
a coating or surface on a biological or non-biological
substrate.
[0088] The radiation employed in the methods of the present
invention may be any radiation effective for the inactivation of
one or more biological contaminants or pathogens of the preparation
of one or more digestive enzymes being treated. The radiation may
be corpuscular, including E-beam radiation. Preferably the
radiation is electromagnetic radiation, including visible light,
infrared, x-radiation, UV light and mixtures of various wavelengths
of electromagnetic radiation. A particularly preferred form of
radiation is gamma radiation.
[0089] According to the methods of the present invention, the
preparation of one or more digestive enzymes to be sterilized is
irradiated with the radiation at a rate effective for the
inactivation of one or more biological contaminants or pathogens of
the preparation. Suitable rates of irradiation may vary depending
upon certain features of the methods of the present invention being
employed, such as the nature and characteristics of the particular
preparation of one or more digestive enzymes being irradiated, the
particular form of radiation involved and/or the particular
biological contaminants or pathogens being inactivated. Suitable
rates of irradiation can be determined empirically by one skilled
in the art. Preferably, the rate of irradiation is constant for the
duration of the sterilization procedure. When this is impractical
or otherwise not desired, a variable or discontinuous irradiation
may be utilized.
[0090] According to the methods of the present invention, the rate
of irradiation may be optimized to produce the most advantageous
combination of product recovery and time required to complete the
operation. Both low (.ltoreq.3 kGy/hour) and high (>3 kGy/hour)
rates may be utilized in the methods described herein to achieve
such results.
[0091] According to a particularly preferred embodiment of the
present invention, the rate of irradiation is not more than about
3.0 kGy/hour, more preferably between about 0.1 kGy/hr. and 3.0
kGy/hr, even more preferably between about 0.25 kGy/hr and 2.0
kGy/hour, still even more preferably between about 0.5 kGy/hr and
1.5 kGy/hr and most preferably between about 0.5 kGy/hr and 1.0
kGy/hr.
[0092] According to another particularly preferred embodiment of
the present invention, the rate of irradiation is at least about
3.0 kGy/hr., more preferably at least about 6 kGy/hr., even more
preferably at least about 16 kGy/hr., and even more preferably at
least about 30 kGy/hr and most preferably at least about 45 kGy/hr
or greater.
[0093] According to the methods of the present invention, the
preparation of one or more digestive enzymes to be sterilized is
irradiated with the radiation for a time effective for the
inactivation of one or more biological contaminants or pathogens of
the preparation of one or more digestive enzymes. Combined with
irradiation rate, the appropriate irradiation time results in the
appropriate dose of irradiation being applied to the preparation of
one or more digestive enzymes. Suitable irradiation times mav vary
depending upon the Particular form and rate of radiation involved,
the nature and characteristics of the particular preparation of one
or more digestive enzymes being irradiated and/or the particular
biological contaminants or pathogens being inactivated. Suitable
irradiation times can be determined empirically by one skilled in
the art.
[0094] According to the methods of the present invention, the
preparation of one or more digestive enzymes to be sterilized is
irradiated with radiation up to a total dose effective for the
inactivation of one or more active biological contaminants or
pathogens in the material, while not producing an unacceptable
level of damage to that material. Suitable total doses of radiation
may vary depending upon certain features of the methods of the
present invention being employed, such as the nature and
characteristics of the particular preparation being irradiated, the
particular form of radiation involved and/or the particular active
biological contaminant or pathogen being inactivated. Suitable
total doses of radiation can be determined empirically by one
skilled in the art. Preferably, the total dose of radiation is at
least 25 kGy, more preferably at least 45 kGy, even more preferably
at least 75 kGy, and still more preferably at least 100 kGy or
greater, such as 150 kGy or 200 kGy or greater.
[0095] The particular geometry of the preparation of one or more
digestive enzymes being irradiated, such as the thickness and
distance from the source of radiation, may be determined
empirically by one skilled in the art.
[0096] According to certain methods of the present invention, an
effective amount of at least one sensitizing compound may
optionally be added to the preparation of one or more digestive
enzymes prior to irradiation, for example to enhance the effect of
the irradiation on the biological contaminant(s) or pathogen(s)
therein, while employing the methods described herein to minimize
the deleterious effects of irradiation upon the preparation of one
or more digestive enzymes. Suitable sensitizers are known to those
skilled in the art, and include, for example, psoralens and their
derivatives and analogs and inactines and their derivatives and
analogs.
[0097] According to the methods of the present invention, the
irradiation of the preparation of one or more digestive enzymes may
occur at any temperature that is not deleterious to the preparation
of one or more digestive enzymes being sterilized. According to one
preferred embodiment, the preparation of one or more digestive
enzymes is irradiated at ambient temperature. According to an
alternate preferred embodiment, the preparation of one or more
digestive enzymes is irradiated at reduced temperature, i.e. a
temperature below ambient temperature, such as 0.degree. C.,
-20.degree. C., -40.degree. C., -60.degree. C., -78.degree. C. or
-196.degree. C. According to this embodiment of the present
invention, the preparation of one or more digestive enzymes is
preferably irradiated at or below the freezing or eutectic point of
the preparation of one or more digestive enzymes. According to
another alternate preferred embodiment, the preparation of one or
more digestive enzymes is irradiated at elevated temperature, i.e.
a temperature above ambient temperature, such as 37.degree. C.,
60.degree. C., 72.degree. C. or 80.degree. C. While not wishing to
be bound by any theory, the use of elevated temperature may enhance
the effect of irradiation on the biological contaminant(s) or
pathogen(s) and therefore allow the use of a lower total dose of
radiation.
[0098] Most preferably, the irradiation of the preparation of one
or more digestive enzymes occurs at a temperature that protects the
preparation from radiation. Suitable temperatures can be determined
empirically by one skilled in the art.
[0099] In certain embodiments of the present invention, the
temperature at which irradiation is performed may be found to lie
within a range, rather than at a specific point. Such a range for
the preferred temperature for the irradiation of a particular
preparation of one or more digestive enzymes may be determined
empirically by one skilled in the art.
[0100] According to the methods of the present invention, the
irradiation of the preparation of one or more digestive enzymes may
occur at any pressure which is not deleterious to the biological
material being sterilized. According to one preferred embodiment,
the preparation of one or more digestive enzymes is irradiated at
elevated pressure. More preferably, the preparation of one or more
digestive enzymes is irradiated at elevated pressure due to the
application of sound waves, the use of a volatile, compression or
other means known to those skilled in the art. While not wishing to
be bound by any theory, the use of elevated pressure may enhance
the effect of irradiation on the biological contaminant(s) or
pathogen(s) and/or enhance the protection afforded by one or more
stabilizers, and therefore allow the use of a lower total dose of
radiation. Suitable pressures can be determined empirically by one
skilled in the art.
[0101] Generally, according to the methods of the present
invention, the pH of the preparation of one or more digestive
enzymes undergoing sterilization is about 7. In some embodiments of
the present invention, however, the preparation of one or more
digestive enzymes may have a pH of less than 7, preferably less
than or equal to 6, more preferably less than or equal to 5, even
more preferably less than or equal to 4, and most preferably less
than or equal to 3. In alternative embodiments of the present
invention, the preparation of one or more digestive enzymes may
have a pH of greater than 7, preferably greater than or equal to 8,
more preferably greater than or equal to 9, even more preferably
greater than or equal to 10, and most preferably greater than or
equal to 11. According to certain embodiments of the present
invention, the pH of the preparation undergoing sterilization is at
or near the isoelectric point of the enzyme(s) contained in the
preparation. According to other embodiments of the present
invention, the pH of the preparation undergoing sterilization is at
or near the pH at which at least one enzyme in the preparation has
maximal affinity for its substrate(s). Suitable pH levels can be
determined empirically by one skilled in the art.
[0102] Similarly, according to the methods of the present
invention, the irradiation of the preparation of one or more
digestive enzymes may occur under any atmosphere that is not
deleterious to the preparation of one or more digestive enzymes
being treated. According to one preferred embodiment, the
preparation of one or more digestive enzymes is held in a low
oxygen atmosphere or an inert atmosphere. When an inert atmosphere
is employed, the atmosphere is preferably composed of a noble gas,
such as helium or argon, more preferably a higher molecular weight
noble gas, and most preferably argon. According to another
preferred embodiment, the preparation of one or more digestive
enzymes is held under vacuum while being irradiated. According to a
particularly preferred embodiment of the present invention, a
preparation of one or more digestive enzymes (lyophilized, liquid
or frozen) is stored under vacuum or an inert atmosphere
(preferably a noble gas, such as helium or argon, more preferably a
higher molecular weight noble gas, and most preferably argon) prior
to irradiation. According to an alternative preferred embodiment of
the present invention, a liquid preparation of one or more
digestive enzymes is held under low pressure, to decrease the
amount of gas, particularly oxygen, dissolved in the liquid, prior
to irradiation, either with or without a prior step of solvent
reduction, such as lyophilization. Such degassing may be performed
using any of the methods known to one skilled in the art.
[0103] In another preferred embodiment, where the preparation of
one or more digestive enzymes contains oxygen or other gases
dissolved within or associated with it, the amount of these gases
within or associated with the preparation may be reduced by any of
the methods and techniques known and available to those skilled in
the art, such as the controlled reduction of pressure within a
container (rigid or flexible) holding the preparation to be treated
or by placing the preparation in a container of approximately equal
volume.
[0104] It will be appreciated that the combination of one or more
of the features described herein may be employed to further
minimize undesirable effects upon the preparation of one or more
digestive enzymes caused by irradiation, while maintaining adequate
effectiveness of the irradiation process on the biological
contaminant(s) or pathogen(s). For example, in addition to the use
of a stabilizer, a particular preparation of one or more digestive
enzymes may also be lyophilized, held at reduced temperature and
kept under vacuum prior to irradiation to further minimize
undesirable effects.
[0105] The sensitivity of a particular biological contaminant or
pathogen to radiation is commonly calculated by determining the
dose necessary to inactivate or kill all but 37% of the agent in a
sample, which is known as the D.sub.37 value. The desirable
components of a preparation of one or more digestive enzymes may
also be considered to have a D.sub.37 value equal to the dose of
radiation required to eliminate all but 37% of their desirable
biological and physiological characteristics.
[0106] In accordance with certain preferred methods of the present
invention, the sterilization of a preparation of one or more
digestive enzymes is conducted under conditions that result-in a
decrease in the D.sub.37 value of the biological contaminant or
pathogen without a concomitant decrease in the D.sub.37 value of
the preparation of one or more digestive enzymes. In accordance
with other preferred methods of the present invention, the
sterilization of a preparation of one or more digestive enzymes is
conducted under conditions that result in an increase in the
D.sub.37 value of the preparation of one or more digestive enzymes.
In accordance with the most preferred methods of the present
invention, the sterilization of a preparation of one or more
digestive enzymes is conducted under conditions that result in a
decrease in the D.sub.37 value of the biological contaminant or
pathogen and a concomitant increase in the D.sub.37 value of the
preparation of one or more digestive enzymes.
EXAMPLES
[0107] The following examples are illustrative, but not limiting,
of the present invention. Other suitable modifications and
adaptations are of the variety normally encountered by those
skilled in the art and are fully within the spirit and scope of the
present invention. Unless otherwise noted, all irradiation was
accomplished using a .sup.6.degree. Co source.
Example 1
[0108] In this experiment, lyophilized trypsin was irradiated (45
kGy at 1.9 kGy/hr) alone or in the presence of a stabilizer (sodium
ascorbate 100 mM) at varying levels of residual solvent
content.
[0109] Method
[0110] 1 ml aliquots of trypsin alone or with 100 mM sodium
ascorbate (10 mg/ml) were placed. in 3 ml vials. Samples were
prepared in triplicate and subjected to lyophilization, either a
primary drying cycle (22 hours, sample temp 0-10.degree. C., shelf
temp 35.degree. C., 10 mT) or a combination of a primary drying
cycle and a secondary drying cycle (60 hours, sample temp
40.degree. C., shelf temp 40.degree. C., 10 mT).
[0111] All samples were resuspended in 1 ml water, and then diluted
1:10 for assay. Assay conditions: 50 units/ml trypsin per
well+BAPNA substrate starting at 3000 .mu.g/ml was serially diluted
3-fold down a 96-well plate. The assay was set up in two 96-well
plates and absorption read at both 405 and 620 nm at 5 and 20
minutes. The absorption at 630 nm (background) was subtracted from
the value at 405 nm to obtain a corrected absorption value. The
change in this value over time between 5 and 15 minutes of reaction
time was plotted and Vmax and Km determined in Sigma Plot using the
hyperbolic rectangular equation).
[0112] Results
[0113] In the absence of stabilizer, lyophilized trypsin exposed to
45 kGy total dose gamma-irradiation showed recovery of 74% of
control activity at the higher residual solvent content level, i.e.
about 2.4% water, and recovery of 85% of control activity at the
lower residual solvent content level, i.e., about 1.8% water.
[0114] In the presence of stabilizer, trypsin exposed to 45 kGy
total dose gamma-irradiation showed recovery of 97% of control
activity at higher residual solvent content levels, i.e. about 3.7%
water, and recovery of 86% of control activity at lower residual
solvent content levels, i.e. about 0.7% water.
[0115] The results of this experiment are shown graphically in
FIGS. 1A-1B.
Example 2
[0116] In this experiment, trypsin was irradiated (45 kGy at 1.6
kGy/hr. and 4.degree. C.) in the presence of a stabilizer (sodium
ascorbate 200 mM) as either a liquid or lyophilized preparation at
varying pH levels.
[0117] Method
[0118] 1 ml of 1 mg/ml (about 3000 IU/ml) trypsin aliquots in the
presence of 35 mM phosphate buffer and 200 mM sodium ascorbate were
made at varying pH levels between 5 and 8.5, inclusive. 400 .mu.l
of each solution was placed in 3 ml vials and then lyophilized and
gamma-irradiated. The remaining portion of each solution was
gamma-irradiated as a liquid. Lyophilized and liquid samples were
assayed at the same time, under the following conditions: Assay
conditions: 5 U/well trypsin (50 U/ml)+BATNA substrate (1 mg/ml)
was serially diluted 3-fold down a 96-well plate. The assay was set
up in two 96-well plates and absorption read at both 405 and 620 nm
at 5 and 20 minutes. The absorption at 630 nm (background) was
subtracted from the value at 405 nm to obtain a corrected
absorption value. The change in this value over time between 5 and
15 minutes of reaction time was plotted and Vmax and Km determined
in Sigma Plot using the hyperbolic rectangular equation).
[0119] Results
[0120] Liquid trypsin samples exposed to 45 kGy total dose
gamma-irradiation showed recovery of between about 70 and 75% of
control activity across the pH range tested. Lyophilized trypsin
samples showed recovery of between about 86 and 97% of control
activity across the same pH ranges. More specifically, the
following results were observed:
1 lyophilized liquid Sample # pH (% of control activity) (% of
control activity) 1 5 91.11 69.87 2 5.5 94.38 74.86 3 6 85.54 75.77
4 6.47 96.26 71.79 5 7 90.40 75.59 6 7.5 96.79 75.63 7 7.8 90.62
74.55 8 8.5 89.59 71.08 The results of this experiment are shown
graphically in FIG. 2.
Example 3
[0121] In this experiment, lyophilized trypsin was irradiated
(42.7-44.8 kGy at 2.65 kGy/hr at 4.degree. C.) alone or in the
presence of a stabilizer (sodium ascorbate 200 mM).
[0122] Method
[0123] 1 ml aliquots of trypsin alone or with 200 mM sodium
ascorbate (1 mg/ml) were placed in 3 ml vials and frozen overnight
at -70.degree. C. Samples were prepared in quadruplicate and
subjected to lyophilization, utilizing primary and secondary drying
cycles (20 hours total).
[0124] All samples were resuspended in 1 ml water, and then diluted
1:10 for assay. Assay conditions: 50 units/ml trypsin per
well+BATNA substrate starting at 3000 .mu.g/ml was serially diluted
3-fold down a 96-well plate. The assay was set up in two 96-well
plates and absorption read at both 405 and 620 nm at 5 and 20
minutes. The absorption at 630 nm (background) was subtracted from
the value at 405 nm to obtain a corrected absorption value. The
change in this value over time between 5 and 15 minutes of reaction
time was plotted and Vmax and Km determined in Sigma Plot using the
hyperbolic rectangular equation).
[0125] Results
[0126] In the absence of stabilizer, lyophilized trypsin exposed to
gamma-irradiation showed recovery of 63% of control activity. In
the presence of stabilizer, lyophilized trypsin exposed to
gamma-irradiation showed recovery of 88% of control activity. The
results of this experiment are shown graphically in FIGS.
3A-3B.
Example 4
[0127] In this experiment, trypsin that had been lyophilized (0.7%
moisture) was irradiated (45 kGy at 1.867 kGy/hr at 3.2.degree. C)
alone or in the presence of a stabilizer (sodium ascorbate 100 mM)
at varying levels of residual solvent content.
[0128] Method
[0129] 1 ml aliquots of trypsin alone or with 100 mM sodium
ascorbate (10 mg/ml) were placed in 3 ml vials and frozen overnight
at -70.degree. C. Samples were prepared in quadruplicate and
subjected to lyophilization (69.5 hours total run time; shelf
temperature 35.degree. C.).
[0130] All samples were resuspended in 1 ml water, and then diluted
1:10 for assay. Assay conditions: 50 units/ml trypsin per
well+BAPNA substrate starting at 3000 .mu.g/ml was serially diluted
3-fold down a 96-well plate. The assay was set up in two 96-well
plates and absorption read at both 405 and 620 nm at 5 and 20
minutes. The absorption at 630 nm (background) was subtracted from
the value at 405 nm to obtain a corrected absorption value. The
change in this value over time between 5 and 15 minutes of reaction
time was plotted and Vmax and Km determined in Sigma Plot using the
hyperbolic rectangular equation).
[0131] Results
[0132] In the absence of stabilizer, trypsin (3.9% water) exposed
to 45 kGy total dose gamma-irradiation showed recovery of 77% of
control activity. In the presence of stabilizer, trypsin (0.7%
water) exposed to 45 kGy total dose gamma-irradiation showed
recovery of 86% of control activity. The results of this experiment
are shown graphically in FIGS. 4A-4B.
Example 5
[0133] In this experiment, lyophilized trypsin was irradiated (45
kGy at 1.9 kGy/hr) alone or in the presence of a stabilizer (sodium
ascorbate 100 mM) at varying levels of residual solvent
content.
[0134] Method
[0135] 1 ml aliquots of trypsin alone or with 100 mM sodium
ascorbate (10 mg/ml) were placed in 3 ml vials. Samples were
prepared in triplicate and subjected to lyophilization, either a
primary drying cycle (25 hours, sample temp 0-10C, shelf temp
35.degree. C., 10 mT) or a combination of a primary drying cycle
and a secondary drying cycle (65 hours, sample temp 40.degree. C.,
shelf temp 40.degree. C., 10 mT).
[0136] All samples were resuspended in 1 ml water, and then diluted
1:10 for assay. Assay conditions: 50 units/ml trypsin per
well+BAPNA substrate starting at 3000 .mu.g/ml was serially diluted
3-fold down a 96-well plate. The assay was set up in two 96-well
plates and absorption read at both 405 and 620 nm at 5 and 20
minutes. The absorption at 630 nm (background) was subtracted from
the value at 405 nm to obtain a corrected absorption value. The
change in this value over time between 5 and 15 minutes of reaction
time was plotted and Vmax and Km determined in Sigma Plot using the
hyperbolic rectangular equation).
[0137] Results
[0138] In the absence of stabilizer, trypsin exposed to 45 kGy
total dose gamma-irradiation showed recovery of 74% of control
activity at the higher residual solvent content level, i.e. about
5.8% water, and recovery of 77% of control activity at the lower
residual solvent content level, i.e., about 5.4% water.
[0139] In the presence of stabilizer, trypsin exposed to 45 kGy
total dose gamma-irradiation showed recovery of 97% of control
activity at higher residual solvent content levels, i.e. about 2.8%
water, and recovery of 90% of control activity at lower residual
solvent content levels, i.e. about 1.1% water.
[0140] The results of this experiment are shown graphically in
FIGS. 5A-5B.
Example 6
[0141] In this experiment, trypsin suspended in polypropylene
glycol 400 was subjected to gamma irradiation at varying levels of
residual solvent (water) content.
[0142] Method Trypsin was suspended in polypropylene glycol 400 at
a concentration of about 20,000 U/ml and divided into multiple
samples. A fixed amount of water (0%, 1%, 2.4%, 4.8%, 7%, 9%, 10%,
20%, 33%) was added to each sample; a 100% water sample was also
prepared which contained no PPG 400.
[0143] Samples were irradiated to a total dose of 45 kGy at a rate
of 1.9 kGy/hr and a temperature of 4.degree. C. Following
irradiation, each sample was centrifuged to pellet the undissolved
trypsin. The PPG/water soluble fraction was removed and the pellets
resuspended in water alone.
[0144] Assay conditions: 5 U/well trypsin (50 U/ml)+BAPNA substrate
(0.5 mg/ml) was serially diluted 3-fold down a 96-well plate. The
assay was set up in two 96-well plates and absorption read at both
405 and 620 nm at 5 and 20 minutes. The absorption at 630 nm
(background) was subtracted from the value at 405 nm to obtain a
corrected absorption value. The change in this value over time
between 5 and 15 minutes of reaction time was plotted and Vmax and
Km determined in Sigma Plot using the hyperbolic rectangular
equation).
[0145] Results
[0146] The irradiated samples containing a mixture of polypropylene
glycol (PPG 400) and water (up to 33% water) retained about 80% of
the activity of an unirradiated trypsin control and activity equal
to that of a dry (lyophilized) trypsin control irradiated under
identical conditions. No activity was detected in the 100% water
sample irradiated to 45 kGy. The results of this experiment are
shown graphically in FIG. 6.
Example 7
[0147] In this experiment, an aqueous solution of trypsin was
subjected to gamma irradiation at varying concentrations of a
stabilizer (sodium ascorbate, alone or in combination with 1.5 mM
uric acid).
[0148] Method
[0149] Trypsin samples (5 Units/sample) were prepared with varying
concentrations of sodium ascorbate, alone or in combination with
1.5 mM uric acid. Samples were irradiated to a total dose of 45 kGy
at a rate of 1.9 kGy/hr and a temperature of 4.degree. C.
[0150] Assay conditions: 5 U/well trypsin (50 U/ml)+50 .mu.l BAPNA
substrate (1 mg/ml). The assay was set up in two 96-well plates and
absorption read at both 405 and 620 nm at 5 and 20 minutes. The
absorption at 630 nm (background) was subtracted from the value at
405 nm to obtain a corrected absorption value. The change in this
value over time between 5 and 15 minutes of reaction time was
plotted and Vmax and Km determined in Sigma Plot using the
hyperbolic rectangular equation).
[0151] Results
[0152] The irradiated samples containing at least 20 mM ascorbate
retained varying levels of trypsin activity compared to an
unirradiated control. Samples containing 125 mM or more ascorbate
retained about 75% of the trypsin activity of an unirradiated
control. Similar results were observed with samples containing
ascorbate in combination with uric acid. The results of this
experiment are shown graphically in FIG. 7.
Example 8
[0153] In this experiment, the protective effect of ascorbate (200
mM) and a combination of ascorbate (200 mM) and Gly-Gly (200 mM) on
two different frozen enzyme preparations (a glycosidase and a
sulfatase) was evaluated.
[0154] Method
[0155] In glass vials, 300 .mu.l total volume containing 300 .mu.g
of enzyme (1 mg/ml) were prepared with either no stabilizer or the
stabilizer of interest. Samples were irradiated with gamma
radiation (45 kGy total dose, dose rate and temperature of 1.616
kGy/hr and -21.5.degree. C. or 5.35 kGy/hr and -21.9.degree. C.)
and then assayed for structural integrity.
[0156] Structural integrity was determined by SDS-PAGE. Three 12.5%
gels were prepared according to the following recipe: 4.2 ml
acrylamide; 2.5 ml 4X-Tris (pH 8.8); 3.3 ml water; 100 .mu.l 10%
APS solution; and 10 .mu.l TEMED. This solution was then placed in
an electrophoresis unit with IX Running Buffer (15.1 g Tris base;
72.0 g glycine; 5.0 g SDS in 1 l water, diluted 5-fold). Irradiated
and control samples (1 mg/ml) were diluted with Sample Buffer
(.+-.beta-ME) in Eppindorf tubes and then centrifuged for several
minutes. 20 .mu.l of each diluted sample (.about.10 .mu.g) were
assayed.
[0157] Results
[0158] Liquid enzyme samples irradiated to 45 kGy in the absence of
a stabilizer showed significant loss of material and evidence of
both aggregation and fragmentation. Much greater recovery of
material was obtained from the irradiated samples containing
ascorbate or a combination of ascorbate and Gly-Gly. The results of
this experiment are shown in FIGS. 8A-8B.
Example 9
[0159] In this experiment, the protective effect of ascorbate (200
mM) and a combination of ascorbate (200 mM) and Gly-Gly (200 mM) on
a frozen glycosidase preparation was evaluated.
[0160] Method
[0161] Samples were prepared in 2 ml glass vials, each containing
52.6 .mu.l of a glycosidase solution (5.7 mg/ml), and either no
stabilizer or a stabilizer of interest, and sufficient water to
make a total sample volume of 300 .mu.l. Samples were irradiated
with gamma radiation (45 kGy total dose, dose rate and temperature
of either 1.616 kGy/hr and -21.5.degree. C. or 5.35 kGy/hr and
-21.9.degree. C.) and then assayed for structural integrity.
[0162] Structural integrity was determined by reverse phase
chromatography. 10 .mu.l of sample were diluted with 90 .mu.l
solvent A and then injected onto an Aquapore RP-300 (c-8) column
(2.1.times.30 mm) mounted in an Applied Biosystems 130A Separation
System Microbore HPLC. Solvent A: 0.1% trifluoroacetic acid;
solvent B: 70% acetonitrile, 30% water, 0.085% trifluoroacetic
acid.
[0163] Results
[0164] Enzyme samples irradiated to 45 kGy in the absence of a
stabilizer showed broadened and reduced peaks. Much greater
recovery of material, as evidenced by significantly less reduction
in peak size compared to control (FIG. 9), was obtained from the
irradiated samples containing ascorbate or a combination of
ascorbate and Gly-Gly.
Example 10
[0165] In this experiment, lyophilized trypsin was irradiated (45
kGy total dose at 1.9 kGy/hr. at 4.degree. C.) in the presence of
Tris buffer (pH 7.6) or phosphate buffer (pH 7.5).
[0166] Method
[0167] Aliquots of a 1000 IU/ml trypsin solution were placed in 3
ml vials and then lyophilized and gamma-irradiated. The remaining
portion of each solution was gamma-irradiated as a liquid. Samples
were assayed under the following conditions: Assay conditions: 5
U/well trypsin (50 U/ml)+BATNA substrate (1 mg/ml) was serially
diluted 3-fold down a 96-well plate. The assay was set up in two
96-well plates and absorption read at both 405 and 620 nm at 5 and
20 minutes. The absorption at 630 nm (background) was subtracted
from the value at 405 nm to obtain a corrected absorption value.
The change in this value over time between 5 and 15 minutes of
reaction time was plotted and Vmax and Km determined in Sigma Plot
using the hyperbolic rectangular equation).
[0168] Results
[0169] Lyophilized trypsin samples exposed to 45 kGy total dose
gamma-irradiation showed recovery of essentially all trypsin
activity in the presence of Tris buffer and sodium ascorbate and
recovery of 88% of trypsin activity in the presence of phosphate
buffer and sodium ascorbate.
Example 11
[0170] In this experiment, lyophilized enzyme preparations (a
glycosidase and a sulfatase) were irradiated in the absence or
presence of a stabilizer (100 mM sodium ascorbate).
[0171] Method
[0172] Glass vials containing 1 mg of enzyme were prepared with
either no stabilizer or 100 mM sodium ascorbate (50 .mu.l of 2M
solution) and sufficient water to make 1 ml of sample. Samples were
lyophilized following moisture levels: glycosidase with stabilizer,
3.4%; glycosidase without stabilizer, 3.2%; sulfate with
stabilizer, 1.8%; and sulfate without stabilizer, 0.7%. Lyophilized
samples were irradiated with gamma radiation (45 kGy total dose at
1.8 kGy/hr and 4.degree. C.) and then assayed for structural
integrity.
[0173] Structural integrity was determined by SDS-PAGE. In an
electrophoresis unit, 6 .mu.g/lane of each sample was run at 120V
on a 7.5%-15% acrylamide gradient gel with a 4.5% acrylamide
stacker under non-reducing conditions.
[0174] Results
[0175] Lyophilized glycosidase samples irradiated to 45 kGy in the
absence of a stabilizer showed significant recovery of intact
enzyme with only some fragmentation. Fragmentation was reduced by
the addition of a stabilizer.
[0176] Similarly, lyophilized sulfatase samples irradiated to 45
kGy in the absence of a stabilizer showed good recovery of intact
enzyme, but with slightly more fragmentation. Fragmentation was
again reduced by the addition of a stabilizer.
[0177] The results of this experiment are shown in FIG. 10.
Example 12
[0178] In this experiment, lyophilized glycosidase preparations
irradiated in the absence or presence of a stabilizer (200 mM
sodium ascorbate or a combination of 200 mM ascorbate and 200 mM
glycylglycine).
[0179] Methods
[0180] Samples were prepared in glass vials, each containing 300
.mu.g of a lyophilized glycosidase and either no stabilizer or a
stabilizer of interest. Samples were irradiated with gamma
radiation to varying total doses (10 kGy, 30 kGy and 50 kGy total
dose, at a rate of 0.6 kGy/hr. and a temperature of -60.degree. C.)
and then assayed for structural integrity using SDS-PAGE.
[0181] Samples were reconstituted with water to a concentration of
1 mg/ml, diluted 1:1 with 2.times. sample buffer (15.0 ml 4.times.
Upper Tris-SDS buffer (pH 6.8); 1.2 g sodium dodecyl sulfate; 6 ml
glycerol; sufficient water to make up 30 ml; either with or without
0.46g dithiothreitol), and then heated at 80.degree. C. for 10
minutes. 10 .mu.l of each sample (containing 5 .mu.g of enzyme)
were loaded into each lane of a 10% polyacrylamide gel and run on
an electrophoresis unit at 125V for about 1.5 hours.
[0182] Results
[0183] About 80% of the enzyme was recovered following irradiation
of the samples containing no stabilizer, with some degradation as
shown in FIGS. 11A-11C. Less degradation was observed in the
samples containing ascorbate alone as the stabilizer, and even less
degradation in the samples containing a combination of ascorbate
and glycylglycine as the stabilizer.
[0184] Having now fully described this invention, it will be
understood to those of ordinary skill in the art that the methods
of the present invention can be carried out with a wide and
equivalent range of conditions, formulations, and other parameters
without departing from the scope of the invention or any
embodiments thereof.
[0185] All patents and publications cited herein are hereby fully
incorporated by reference in their entirety. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that such publication is
prior art or that the present invention is not entitled to antedate
such publication by virtue of prior invention.
* * * * *